This invention relates to a method of enhancing mutant enzyme activities in lysosomal storage disorders by administration of competitive inhibitors of the enzymes, in particular, imino sugars and related compounds.
2. Background Information
Proteins are synthesized in the cytoplasm, and the newly synthesized proteins are secreted into the lumen of the endoplasmic reticulum (ER) in a largely unfolded state. In general, protein folding is an event governed by a principle of self assembly. The tendency of proteins to fold into their native (active) conformation is contained in their amino acid sequences (1). In vitro, the primary structure folds into secondary structres of a-helices and b-sheets coupled with hydrophobic collapse in the formation of biologically active tertiary structure which also gains increased conformational stability. However, the folding of protein in vivo is rather complicated, because the combination of ambient temperature and high protein concentration stimulates the process of aggregation, in which amino acids normally buried in the hydrophobic core interact with their neighbors non-specifically. To avoid this problem, protein folding is usually facilitated by a special group of proteins called molecular chaperones which prevent nascent polypeptide chains from aggregating, and bind to protein so that the protein refolds in the active state (2).
Molecular chaperones are present in virtually all types of cells and in most cellular compartments. Some are involved in the transport of proteins and in supporting cells to survive under stresses such as heat shock and glucose starvation. Among the molecular chaperones (3-6), BIP (immunoglobulin heavy-chain binding protein, Grp78) is the best characterized chaperone of the ER (7). Like other molecular chaperones, BIP interacts with many secretory and membrane proteins within the ER throughout their maturation, although the interaction is normally weak and short-lived when the folding proceeds smoothly. Once the native protein conformation is achieved, the molecular chaperone no longer binds. However, the interaction between BIP and those proteins that fail to fold, assemble or be properly glycosylated becomes stable, and usually leads to degradation of these proteins through the ubiquitin pathway. This process serves as a xe2x80x9cquality controlxe2x80x9d system in the ER which ensures that only properly folded and assembled proteins are transported to the Golgi complex for further maturation, and those improperly folded proteins are retained for subsequent degradation (8).
In many hereditary disorders, mutant gene products are structurally altered and may not fold correctly, signalling the quality control system to retain and degrade them in situ. This process may contribute significantly to the protein deficiency, although the function of the protein may have been only partially impaired (9-12). For example, the most common mutation in cystic fibrosis, a deletion of phenylalanine-508 (xcex94F508) in the CFTR protein which functions as a chloride channel in the plasma membrane, results in misfolding and retardation of the xcex94F508-CFTR protein in the ER, and subsequent degradation by the cytosolie proteasome system (13-14), even though it retains almost full biologic activity when inserted into plasma membranes (15) The list of diseases caused by mutations that alter protein folding is increasing, and it includes xcex11-antitrypsin deficiency (16-17), familial hypercholesterolemia (18), Alzheimer""s disease (18a), Marfan syndrome (19), osteogenesis imperfecta (20), carbohydrate-deficient glycoprotein syndrome (21), and Maroteaux-Lamy syndrome (22).
Lysosomal storage disorders are a group of diseases resulting from the abnormal metabolism of various substrates, including glycosphingolipids, glycogen, mucopolysaccharides and glycoproteins. The metabolism of exo-and endogenous high molecular weight compounds normally occurs in the lysosomes, and the process is normally regulated in a stepwise process by degradation enzymes. Therefore, a deficient activity in one enzyme may impair the process, resulting in an accumulation of particular substrates. Most of these diseases can be clinically classified into subtypes: i) infantile-onset; ii) juvenile-onset; or iii) late-onset. The infantile-onset forms are often the most severe usually with no residual enzyme activity. The later-onset forms are often milder with low, but often detectable residual enzyme activity. The severity of the juvenile-onset forms are in between the infantile-onset and late-onset forms. Table 1 contains a list of a number of known lysosomal storage disorders and their associated defective enzymes. In the adult-onset forms of lysosomal storage disorders listed in Table 1, certain mutations cause instability of the encoded protein.
In their earlier filed patent application (U.S. application Ser. No. 09/087,804), the present inventors proposed a novel therapeutic strategy for Fabry disease, a lysosomal storage disorder caused by deficient lysosomal xcex1-galactosidase A xcex1-Gal A) activity in which certain mutations encoded mutant proteins which have folding defects. The application presented evidence demonstrating that 1-deoxygalactonojirimycin (DGJ), a potent competitive inhibitor of (xcex1-Gal A, effectively increased in vitro stability of a mutant xcex1-Gal A (R301Q) at neutral pH and enhanced the mutant enzyme activity in lymphoblasts established from Fabry patients with the R301Q or Q279E mutations. Furthermore, oral administration of DGJ to trangenic mice overexpressing a mutant (R301Q) (xcex1-Gal A substantially elevated the enzyme activity in major organs (24).
The principle of this strategy is as follows. Since the mutant enzyme protein appears to fold improperly in the ER where pH is neutral, as evidenced by its instability at pH 7 in vitro (25), the enzyme protein would be retarded in the normal transport pathway (ERxe2x86x92Golgi apparatusxe2x86x92endosomexe2x86x92lysosome) and subjected to rapid degradation. In contrast, an enzyme protein with a proper folding conformation could be efficiently transported to the lysosomes and remain active, because the enzyme is more stable below pH 5. Therefore, a functional compound which is able to induce a stable molecular conformation of the enzyme is expected to serve as a xe2x80x9cchemical chaperonexe2x80x9d for the mutant protein to stabilize the mutant protein in a proper conformation for transport to the lysosomes. Some inhibitors of an enzyme are known to occupy the catalytic center of enzyme, resulting in stabilization of its conformation in vitro, they may also serve as xe2x80x9cchemical chaperonesxe2x80x9d to enforce the proper folding of enzyme in vivo, thus rescue the mutant enzyme from the ER quality control system. It is noted that while this is believed to be the mechanism of operation of the present invention, the success of the invention is not dependent upon this being the correct mechanism.
The present inventors have unexpectedly found that potent competitive inhibitors for enzymes associated with lysosomal storage disorders enhance the activity of such enzymes in cells when administered at concentrations lower than that normally required to inhibit the intracellular enzyme activity. The effect is particularly significant on certain defective or mutant enzymes, but also occurs in cells containing the normal enzyme type.
Accordingly, it is one object of the present invention to provide a method of preventing degradation of mutant enzymes associated with lysosomal storage diseases in mammalian cells, particularly in human cells.
It is a further object of the invention to provide a method of enhancing the activity of enzymes associated with lysosomal storage disease in mammalian cells, particularly in human cells. The method of the present invention enhance the activity of both normal and mutant xcex1-Gal A, particularly of mutant xcex1-Gal A which is present in certain forms of Fabry disease. The methods of the present invention also enhance the activity of certain mutant xcex2-galactosidase and glucocerebrosidase and are expected to be useful in other lysosomal storage diseases, including those listed in Table 1.
In addition, the methods of the invention are also expected to be useful in nonmammalian cells, such as, for example, cultured insect cells and CHO cells which are used for production of xcex1-Gal A for enzyme replacement therapy.
It is yet a further object of the invention to provide a method of treatment for patients with lysosomal storage disorders such as those listed in Table 1.
Compounds expected to be particularly effective for Fabry disease in the methods of the invention arc galactose and glucose derivatives having a nitrogen replacing the oxygen in the ring, preferably galactose derivatives such as 1-deoxygalatonojirimycin and 4-epr-xcex1homonojirimycin. The term xe2x80x9cgalactose derivativexe2x80x9d is intended to mean that the hydroxyl group at the C-3 position is equatorial and the hydroxyl group at the C-4 position is axial, as represented. For example, by the following structures 
wherein R0 represents H, methyl or ethyl; R1 and R1xe2x80x2 independently represent H, OH, a 1-4 carbon alkyl alkoxy or hydroxyalkyl group (e.g., CH2OH); R2 and R2xe2x80x2 independently represent H, OH or alkyl group (n=1xe2x88x928).
Other specific competitive inhibitors for xcex1-galactosidase. such as for example, calystegine A3 and B2, and N-methyl derivatives of these compounds should be useful in the method of the invention. The calystegine compounds can be represented by the formula 
wherein for calystegine A3: R0=H, R2=R2xe2x80x2=H. R4=OH. R4xe2x80x2=R7=H; for calystegine B2: R0=H. R2=OH, R2xe2x80x2=R4xe2x80x2=H, R4=OH, R7=H; for N-methyl-calystegine A3: R0=CH3, R2=R2xe2x80x2=H. R4=OH, R4xe2x80x2=R7=H; for N-methyl-calystegine B2: R0=CH3R2=OH, R2xe2x80x2=R4xe2x80x2=H. R4=OH. R7=T.
Administration of a pharmaceutically effective amount of a compound of formula 
wherein R0 represents H, methyl or ethyl; R1 and R1xe2x80x2independently represent H, OH, a 1-4 carbon alkyl, alkoxy or hydroxyalkyl group (e.g. CH2OH): R2 and R2xe2x80x2 independently represent H, OH or alkyl group (n=1xe2x88x928); R4 and R4xe2x80x2 independently represent H, OH; or a compound selected from the group consisting of xcex1-allo-homonojirimycin, xcex1-galaco-homonojirimycin, xcex2-1-C-butyl-deoxygalactonojirimycin, calystegine A3,calystegine B2 and their N-alkyl derivatives will alleviate the symptoms of Fabry disease by increasing the residual enzyme activity in patients suffering from Fabry disease.
Compounds expected to be particularly effective for GMI-gangliosidosis in the methods of the invention are galactose derivatives having a nitrogen replacing the oxygen in the ring or a nitrogen at the same position of the anomeric position of a pyranose ring, preferably galactose derivatives such as 4-epi-isofagoimine and 1-deoxygalactonojirimycin.
Administration of a pharmaceutically effective amount of a compound of formula 
wherein R0 represents H, methyl or ethyl: R1 and R1xe2x80x2 independently represent H, OH, a 1-4 carbon alkyl, alkoxy or hydroxyalkyl group (e.g. CH2OH), R2 and R2xe2x80x2 independently represent H. OH or alkyl group (n=1xe2x88x928): or a compound selected from the group consisting of 4-epi-isofagomine, and 1-deoxygalactonojirimycin and their N-alkyl derivatives will alleviate the symptoms of GMI-gangliosidosis by increasing the residual xcex2-galactosidase activity in patients suffering from GMI-gangliosidosis.
Compounds expected to he particularly effective for Gaucher disease in the methods of the invention are glucose derivatives having a nitrogen replacing the oxygen in the ring or a nitrogen at the same position of the anomeric position of a pyranose ring preferably glucose derivatives such as N-dodecyl-deoxynojirimycin and isofagomine. The term xe2x80x9cglucose derivativexe2x80x9d is intended to mean that the hydroxyl groups at the C-3 and C-4 positions are equatorial as represented, for example, by the following structures: 
wherein R0 represents H alkyl chain (n=8xe2x88x9212); R0xe2x80x2 represents H, a straight chain or branched saturated or unsaturated carbon chain containing 1-12 carbon atoms, optionally substituted with a phenyl, hydroxyl or cyclohexyl group; R1 and R1xe2x80x2 independently represent H, OH, a 1-4 carbon alkyl, alkoxy or hydroxyalkyl group (e.g., CH2OH), R2 and R2xe2x80x2 independently represent H, OH or alkyl group (n=1xe2x88x928).
Other specific competitive inhibitors for xcex2-glucosidase such as for example, calystegine A3, B1, B2 and C1 and their derivatives of these compounds should be useful in the method of the invention. The calystegine compounds can be represented by the formula 
wherein for calystegine A3: R0=H, R2=R2xe2x80x2H, R4=OH, R4xe2x80x2=R7H; for calystegine B1: R0=H, R2=R2xe2x80x2=R4xe2x80x2=H, R4=OH, R7=OH; for calystegine B2; R0=H, R2=OH. R2xe2x80x2=R4xe2x80x2=H. R4=OH. R7=H; for calystegine C1: R0=H, R2=H, R2xe2x80x2=H, R4=H, R4xe2x80x2=H, R4=OH.
Administration of a pharmaceutically effective amount of a compound of formula 
wherein R0 represents H, alkyl chain (n=8xe2x88x9212); R0xe2x80x2 represents H, a straight chain or branched saturated or unsaturated carbon chain containing 1-12 carbon atoms, optionally substituted with a phenyl, hydroxyl, or cyclohexyl group; R1 and R1xe2x80x2 independently represent H, OH, a 1-4 carbon alkoxy or hydroxyalky group (e.g. CH2OH); R2 and R2xe2x80x2 independently represent H, OH or alkyl group (n=1xe2x88x928): or a compound selected from the group consisting of isofagomine, N-butyl-isofagomine, N-(3-cyclohexylpropyl)-isofagomine, N-(3-phenylpropyl)-isofagomine and N-[(2E, 6Z, 10Z)-3,7,11-trimethyldodecatrenyl]-isofagomine. N-dodecyl-deoxynojirimycin will alleviate the symptoms of Gaucher disease by increasing the residual glucocerebrosidase activity in patients suffering from Gaucher disease. Other competitive inhibitors of glucerebrosidase, such as calystegine compounds and N-alkyl derivatives thereof should also be useful for treating Gaucher disease. Similarly, known competitive inhibitors of other enzymes associated with lysosomal storage disorders listed in Table 1 will be useful in treating those disorders.
Persons of skill in the art will understand that an effective amount of the compounds used in the methods of the invention can be determined by routine experiments, but is expected to be an amount resulting in serum levels between 0.01 and 100 xcexcM, preferably between 0.01 and 10 xcexcM, most preferably between 0.05 and 1 xcexcM. The effective dose of the compounds is expected to be between 0.5 and 1000 mg/kg body weight/day, preferably between 0.5 and 100 mg/kg body weight/day, most preferably between 1 and 50 mg/kg body weight/day. The compounds can be administered alone or optionally along with pharmaceutically acceptable carriers and excipients, in preformulated dosages. The administration of an affective amount of the compound will result in an increase in the lysosomal enzymatic activity of the cells of a patient sufficient to improve the symptoms of the disease.
In many lysosomal storage diseases, much of the clinical variability and age of onset can be attributed to small differences in the residual activity of the affected enzyme (25a). Pseudodeficiency of lysosomal storage disorders identified as clinically healthy probands with severely reduced activity (10-20% of normal) of a lysosomal enzyme suggests that a small increase of residual enzyme activity could have a large effect on the disease (25b). Particularly in Fabry disease, a small augmentation in enzyme stability resulting in an increase of residual xcex1Gal A activity is expected to have a significant impact on the disease, based on the observations on the cardiac variants with 10% residual activity (2). Therefore, a small percentage increase of the residual enzyme activity may alleviate symptoms of the disease or significant delay the development of the disease.
Compounds disclosed herein and other competitive inhibitors of enzymes associated with lysosomal storage diseases which will be known to those of skill in the art will be useful according to the invention in methods of enhancing the intracellular activity of normal and mutant enzymes associated with such disorders and treating the disorders.